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Chapter 2

Social and Reproductive Behavior in the Madagascan Poison , laevigata, with Comparisons to the Dendrobatids

Abstract

I present the first behavioral study of natural populations of a Madagascan poison frog. Focal watches of marked individuals were conducted for 925 hours, in five populations, across two seasons. Like the New World dendrobatids, these diurnal anurans eat ants and are aposematically colored. Data are presented that provide additional instances of convergence with the dendrobatids, including 1) extended male-male fights over defended resources necessary for reproductive success of both sexes, 2) stereotyped, highly tactile courtships in which the female may reject initial oviposition sites, and 3) complex maternal care. Females return to water-filled phytotelmata, or wells, and lay trophic eggs for their tadpoles. Mantella laevigata has the minimum possible clutch size in anurans— usually one—suggesting a high degree of parental investment. Males defend wells, which attract females who oviposit in the wells. Fertilized eggs may hatch and metamorphose, or may be eaten by a tadpole already in that well, of which the territorial male is probably the father. Unfertilized eggs serve as food for tadpoles. Oviposition-site scouting behavior of both sexes, and the dependency of tadpole presence on the position of eggs laid, provide evidence of

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context-dependent, and assessment, behavior. Females leave courtships most often only after visiting potential oviposition sites, while males usually leave to engage other males in aggression, suggesting that territory maintenance may be the most important component of male reproductive success. Two other species of often prevent M. laevigata from using defended oviposition sites, and larval crane-flies predate the eggs of all frog species using water-filled wells.

Introduction

The poison frogs of Madagascar, genus Mantella, have long been of interest to students of anuran behavior, largely because there are many similarities between species of Mantella and the toxic members of the neotropical family

Dendrobatidae, the poison-dart frogs. Many species of dendrobatids, especially those in the genera Dendrobates (sensu lato) and Phyllobates, are known for their territoriality, stereotyped courtship sequences, and complex parental care. Until now, nothing was known of the social system of any species of Mantella. This study marks the first behavioral research conducted on Mantella in the field.

When Boulenger identified Mantella in 1882, he placed the genus in

Dendrobatidae. Although he later formalized this taxonomic placement on the basis of their shared lack of teeth (Boulenger 1914), it is clear that he felt that the two groups simply shared a gestalt (Boulenger 1882). Though Boulenger did not know it, the aposematic coloration in both groups arises, in part, from identical classes of lipophilic alkaloids in the skin, including pumiliotoxins and

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decahydroquinolines (Daly et al. 1996). Among anurans, these classes of toxins are otherwise known only in two other (unrelated) frog genera [Melanophryniscus

(Bufonidae) and Pseudophryne (Myobatrachidae), Daly et al. 1984]. Other similarities between Mantella and the dendrobatids include small size (most species have a snout-vent length of 20-30 mm), diurnality, terrestrial and/or arboreal habit, and a diet consisting primarily of ants and mites. Captive dendrobatids lose their toxicity (Daly et al. 1980; Daly et al. 1992), as do captive

Mantella (Daly et al. 1997), suggesting that the building blocks for the alkaloids are dietary. A high percentage of ants in the diet of dendrobatids is correlated with toxicity (Caldwell 1996), and some ants (genus Solenopsis) in the dendrobatid diet produce some of the simpler alkaloids themselves (Spande et al. 1998; Jones et al. 1999).

Mantella laevigata, commonly known as the climbing Mantella, has more characters in common with the aposematic dendrobatids than do the other ten species of Mantella. It is not fully terrestrial, and has expanded toe-pads. It also breeds in water-filled tree holes or broken bamboo (phytotelmata), much like the preferred bromeliads of some dendrobatids. Amplexus is neither reliably axillary nor inguinal in M. laevigata, which are the two most common mating positions in anurans; M. laevigata adopts a variety of positions during mating amplexus (pers. obs.). In several dendrobatids, amplexus is also unlike that of most anurans, being absent (Crump 1972; Limerick 1980), or cephalic, a highly derived form in which the male grasps the female by the head (Myers et al. 1978;

Wells 1980). Finally, the larval morphology of M. laevigata resembles that of the oophagous dendrobatid tadpoles, having reduced labial denticles and a large, keratinized horny beak (Glaw & Vences 1994).

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Several other taxa are known from both South America and Madagascar, but not Africa (e.g., side-neck turtles, Underwood 1976; iguanians, Frost & Etheridge

1989; boas, Kluge 1991). These examples are consistent with reconstructions of

Gondwana that suggest that Africa and South America partially broke from the rest of the Southern landmass by the late Jurassic, and that by the early

Cretaceous, Africa and South America were rifting, leaving a more recent and direct land connection between South America and Madagascar than between

Africa and Madagascar (Scotese & Golonka 1992). These observations might lead to a hypothesis of sister-group relationship between the dendrobatids and

Mantella, but phylogenetic analyses, though not complete with regard to the taxa investigated, provide evidence against such a historical hypothesis (e.g. Hay et al. 1995). Thus, it seems that all similar specializations between the dendrobatids and Mantella are independently evolved.

While a dendrobatid-Mantella relationship would support the hypothesis of

Gondwana reconstruction outlined above, it provides little insight into more general causal chains that may have led to what we observe today. Complex convergences, involving distinct processes, can help identify environmental factors that might lead to similar results in the different organisms. That is, the known existence of several convergences may suggest that others are present.

Based on the known similarities described above, my goal was to discover the social system and natural history of M. laevigata, with the broad hypothesis that some or all of the behaviors of aposematic dendrobatids would be present in M. laevigata. I predicted that male M. laevigata would be territorial, defending calling and/or oviposition sites from other males, as several dendrobatids do

(e.g., D. granuliferus, Crump 1972; D. pumilio, Bunnell 1973; E. femoralis,

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Roithmair 1992). I predicted that M. laevigata would have a stereotyped courtship sequence similar to that of some dendrobatids, in which a male attracts a female using advertisement calls, changes to a softer courtship call, and through repeated tactile interaction with the female leads her to one or more potential oviposition sites until she accepts one, or abandons the courtship (see review by Wells 1977; also Colostethus stepheni and C. marchesianus, Juncá 1998). I also predicted that M. laevigata would have complex maternal care of the type found in the oophagous dendrobatids, in which mothers return to individual tadpoles repeatedly to feed them trophic (unfertilized) eggs (see reviews by

Weygoldt 1987; Crump 1996). This behavior has also been observed in three unrelated species of anurans, two hylids (Osteopilus brunneus, Lannoo et al. 1986; and Anotheca spinosa, Jungfer 1996; Thompson 1996), and one rhacophorid

(Chirixalus eiffingeri, Ueda 1986; Kam et al. 1996).

In my 1999 field season, I had already failed to falsify these broadly defined

"hypotheses of convergence,” based on the behavior I had observed in M. laevigata in 1997. I then focused on quantifying what variables affect the outcomes of territorial encounters and courtships, and what and how often other organisms in the phytotelmata (well) community interact with M. laevigata. With these data it is possible to construct a coherent picture of the selective pressures affecting reproductive success for these frogs.

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Methods

Mantella laevigata was observed during the Malagasy summers of 1997 and

1999 (January through May, and January through April, respectively). The research was conducted on the 510 hectare island of Nosy Mangabe, which lies at the northern end of the Bay of Antongil, five kilometers south of the town of

Maroantsetra, Toamasina Province, in northeastern Madagascar (15º 30’ S, 49º 46’

E). Nosy Mangabe is a “Special Reserve” in the Masoala National Park consisting mainly of 100 – 400 year old secondary forest. There are two summits, the highest being 332 m, and most of the island is inaccessible by trail. M. laevigata are found throughout the island, but are densest in coastal bamboo stands. The bamboo on Nosy Mangabe--Bambusa vulgaris--was introduced, but in Marojezy, the other region in which M. laevigata is found, the frogs are found in association with native bamboo species, including Ochlandra capitata.

The goals of the 1997 and 1999 seasons were distinct. Prior to my arrival in

1997, no researchers had observed these frogs in the wild for more than a few hours. As such, my knowledge of the system was based entirely on what I learned anew every day. I therefore made observations in 1997 on an ad libitum basis, in which I changed "focal" any time a more interesting behavior was observed elsewhere, in order to maximize knowledge about all aspects of the social system of these frogs. In 1999, the intent was to quantify behaviors identified during 1997. The descriptions of behaviors throughout this paper are based on two seasons of observations, while the quantifiable data—e.g., analysis

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of what percentages of behaviors ended in particular ways—are based entirely on 1999 data.

I conducted ad libitum sampling with continuous recording (Martin & Bateson

1993) on populations of marked frogs in coastal bamboo stands in 1997, while in

1999, I used focal watches with continuous recording (Altmann 1974). Three of the four populations watched in 1997 were in three adjacent bamboo stands, encompassing a total area of approximately 1500 m2, though the area taken up by bamboo amounts to only 147 m2 (stands 4, 5, and 6; Fig. 1). Some females traveled among these stands, but males were never observed to do so. The fourth population (bamboo stand 3) is 2.8 km away, and no movement of frogs was recorded between that and the other focal populations. The two populations watched in 1999 were in stands 650 meters apart (stands 2 and 4; Fig.

1), and no movement of animals was observed between these populations.

In 1997, I and an assistant (JM) observed marked individuals for 509 hours.

Animals were caught by hand, and by using small aquarium dipnets. All animals were toe-clipped, in addition to being given a mark which should have enabled us to identify the animals from a distance, including tying small beaded waistbands onto them, and stitching beads directly into their dorsums. None of the marks given in 1997 was reliable for more than two weeks. In 1999, I and a second assistant (GF) conducted 416 hours of focal watches on individuals tattooed on their dorsums with a portable battery-operated tattoo machine

(Dermo-Marker Tattoo Machine model 401). These tattoos required re-inking every four-six weeks, but the animals returned to their territories and resumed calling and territorial defense (if male) immediately upon being returned from tattooing, so the marking technique did not appear to interfere with behavior. In

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both 1997 and 1999, I recorded mass and snout-vent length (SVL) for all marked animals (N=218 in 1997, N=83 in 1999; total N=301).

We conducted focal watches in 1999 in half hour intervals. We watched any given for as much as two hours per day, but attempts were made to diversify the animals watched. Dominant, territorial males were the easiest to find, so they were watched more often than other individuals. Individuals who disappeared under the leaf litter or up bamboo during a focal watch were searched for until the next fifteen minute interval, at which point a new focal animal was found.

The most common behavior observed and recorded was courtship.

Courtship bouts are defined as uninterrupted courtship segments, during which neither participant engages with a third individual, and those courting are within 1 meter of each other. When courtships are interrupted, they may resume later. If the same individuals began courting again within fifteen minutes of a courtship bout being interrupted, the ensuing courtship was coded as another bout in the same courtship. The sum total of bouts within one courtship is referred to as a complete courtship.

At the beginning of every day of focal watching, we examined the contents of all water-filled wells in the vicinity of the population with a mini-maglite, and M. laevigata eggs and tadpoles, heterospecific frogs and broods, and egg predators were censused. These observations were repeated after any frog activity was seen around a given well.

During the 1997 season, 87.8% of the focal watch hours were conducted in the morning, primarily between 5:30 and 10:00 am, when the frogs are most active.

In 1999, 95.3% of focal watches were conducted in the morning. During 416

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hours of focal watches in 1999, 274.5 hours (70.0%) were spent watching adult males, 101 hours (24.3%) were spent watching adult females, and the remaining hours spent watching juveniles or adults of unknown sex.

Statistics

Fisher's Exact Test was used to analyze the difference in oviposition location between courtships and incidents of maternal care. Individual females were not clustered in the analysis presented (which would control for the effect of individuals tending to make similar choices repeatedly), but other analyses

(including contingency tables using Rao and Scott correction for clustered data) revealed similar significance levels when controlled for non-independence between individuals. Chi Square goodness of fit tests were used to analyze male discrimination of wells, in which independent observations of the contents of wells in the population were used to calculate "expected" values, and the contents of wells to which males took females are the "observed" values. Again, alternate analyses, in which the same males in multiple courtships were treated as non-independent events, yielded similar results (calculated a 95% confidence interval for the ratio, using clustering). Logistic regressions with cluster analyses were used to address the significance of much of the data in which the same animal was engaged in multiple incidences of the measured behavior, including the analysis of female discrimination of wells, with repeated, individual females clustered as non-independent variables. The results presented, therefore, are as conservative as is possible with the existing data.

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All of the data described in the preceding paragraph were also analyzed using resampling techniques, which make no a priori assumptions regarding the distribution of the data. The p-values obtained from resampling statistics were all as or more significant than those generated using more traditional tests.

Results

Descriptions of Three Behaviors (from 1997 & 1999 observations)

Territorial Behavior

During 925 hours of observations, 215 fights were witnessed between males, in 367 bouts. Fight bouts between males last from ten seconds to more than 1.5 hours. Fights take place primarily over territories, the best of which include water-filled wells in which mating takes place, and eggs and tadpoles develop.

Wells are the limiting resource for these frogs (see chapter three). Males defend these wells, and an area not less than 500 cm diameter around them, with advertisement calls and fights.

Males use two distinct forms of territorial defense, and an additional category of male does not defend territories at all. Among the territorial males, some defend resources, other appear not to. I define males in possession of territories with wells in them “resource-defense territorial,” or RD-territorial. I define males in possession of territories without wells as “no-resource territorial", or

NR-territorial. As wells are known to be limiting, and other parameters such as

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food are not, territories without wells in them are defined as without resource, and therefore of low quality. Both NR-territorial and RD-territorial males scout in other males’ territories, usually without advertisement calls, when they are not defending their own territory. Finally, there are "aterritorial" males that have not been seen defending any area, but that spend time in territorial males’ territories and try to attract females therein. These "aterritorial" males may in fact be NR- territorial, with defended territories outside the range of my observations. When either NR-territorial or aterritorial males are in territories with wells, I refer to them as non-residents, and the territory-holder as the resident.

Both NR-territorial and aterritorial males sometimes successfully lead females into matings in wells in territories not their own. Often these non-resident males are seen by the resident (territory-holder) and attacked, at which point they respond in one of two ways. Non-resident males may fight back, often repeatedly (several bouts); or they may be docile, not call, and submit to being amplexed by the male resident. Resident males do not amplex, or try to court, non-responsive animals of either sex for more than five minutes, and leave the non-resident after a short time.

RD-territorial males can usually be found in a given territory, not larger than

2 meters square, which contains at least one water-filled well—usually broken bamboo. RD-territorial males call frequently from their territories. NR-territorial males have a high rate of site fidelity as well, but the sites they call from do not have wells in them. These males call and attract females to these low-quality territories, after which they lead females into other males’ (high-quality) territories. Territory borders, and even interiors, are repeatedly fought over by the same males. Fights include male-male amplexus, belly-to-belly wrestling,

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tumbling, leaping on to one another, and chasing. Aterritorial males have home ranges that they do not defend against other males.

Courtship Behavior

During 925 hours of observations, 348 courtships were witnessed in 521 bouts. Courtships involve stereotyped sequences that begin when the male gives an advertisement call, often from a raised perch, such as a palm axil or the well he defends, and a female responds by approaching. When a male sees a female approaching, he moves towards her and makes initial contact by putting his throat either on the top of her head or on her dorsum (behavior hereafter referred to as "chinning"). The male often faces the female during chinning, and less often has his body perpendicular to hers (see Fig. 2). His vocalizations change at this point from the louder, two-note advertisement call to the softer, often single- note courtship call. The male chins the female for up to five minutes before beginning to lead her to a potential oviposition site. He hops not more than 10 centimeters in front of the female, emitting courtship calls, sporadically returning to chin her again. During some courtships, the male alternates chinning of the female with non-reproductive axillary amplexus. For up to 30 minutes, the male leads the female to a potential oviposition site, then climbs to the top of it, and chins her again before allowing her to enter. She then enters, goes into the water, and the male follows, emitting continuous courtship vocalizations. If she accepts the well by remaining inside, the male amplexes her on the inside of the well, and a single egg is laid (see clutch size below). This is the first time that amplexus occurs during the courtship. If she rejects the well, she hops out while

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the male calls from inside the well. In most cases, he then follows and catches her, chins her again, and begins the repeated chinning, leading and calling while leading her to another potential oviposition site.

Only two pairs of M. laevigata were observed in reproductive amplexus without first engaging in the stereotyped courtship sequence. These two exceptions occurred when the female entered the well and began ovipositing at the water line, at which point the male followed her in and amplexed her, and an egg was laid. In both cases, the egg was quickly eaten by the resident tadpole, suggesting attempted maternal care (see below) rather than true courtship.

Some would-be courtships turn into fights, when silent aterritorial or NR- territorial males enter a calling RD-territorial male's territory, and are chinned by the resident male. It is difficult for humans, and perhaps frogs, to distinguish between the sexes without behavioral cues. This is true despite statistically significant sexual dimorphism for both SVL and mass in this species (N=44 females, 70 males; Range, Mean ± SD: female SVL (mm): 25.0 – 31.0, 28.06 ± 0.18; male SVL (mm) 24.0 – 30.0, 26.79 ± 0.14; female mass (g): 1.15 – 2.05, 1.72 ± 0.03; male mass (g): 1.10 – 1.90, 1.53 ± 0.02. Independent Samples T-test, t=5.54 for mass, t=5.50 for SVL, p<0.0001 for both variables).

Females were often observed in scouting behavior, in which they investigate several wells in succession, climb them, look in, and go into the water. While scouting, females ignore the advertisement calls of nearby males, and retreat when males approach them. Males using all of the three territorial categories were also seen scouting potential oviposition sites, never calling while doing so.

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Maternal Care

During 925 hours of observations, 10 incidences of maternal care of tadpoles were witnessed. Females were observed climbing wells alone, ignoring the advertisement calls of nearby males, backing into these wells, and depositing single eggs at or below the water line. In all ten cases, a resident tadpole was observed eating the egg within 30 minutes of deposition. In five of these cases, the females who returned alone had previously been seen in mating amplexus with males in those same wells. I do not, however, have more direct evidence that the tadpoles in these wells belonged to these females, as viable wells often have eggs from several ovipositions and from different mating pairs, at the same time.

Furthermore, two of the successful “courtships” observed appear to have been attempted maternal care in which a territorial male followed the female into the well and amplexed her while she was already positioned at the water line, which is otherwise never seen in mating pairs. These eggs were also promptly eaten by tadpoles. On five occasions, an egg deposited by a single or amplexed female was completely gone within five minutes of the female or mating pair depositing the egg and leaving the well. This suggests that both maternal care and oophagy generally may be more common than these data suggest, as the evidence is quickly erased, rendering the behavior invisible to the researcher.

Tadpoles were never observed eating anything but conspecific eggs, and wells with tadpoles in them were not free of mosquito or other small arthropod larvae or detritus.

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Quantification of Observed Behaviors, from 1999 Data

Resident territorial males fought non-residents in 82.4% of total fights observed (total n=131), of which the residents won 88.9%. Of the remaining

17.6% of fights (n=23), 9 maintained territory boundaries between two resident males, and 14 were fought between non-resident males when the resident was absent. Individual males were predictably winners or losers in successive fights.

Eleven males were observed to participate in five or more fight bouts (range: 7-

45), of which five won less than 23% of fights (range: 0 - 22.2%), while the other six won more than 69% of fights (range: 69.2 - 85.7%). In addition, an initiator's advantage was apparent, as only 17 of 131 complete fights (12.9%) were won by non-initiators, 10 of which were won by residents who were attacked by non- residents.

Apparently, territory is the primary reason for fighting. Ninety-six of 131 fight bouts were fought over territory, in which no female was within 1 meter of either male participant; 31 fight bouts were fought over territory while a female was within 1 meter of the males; and 4 fights appeared to be entirely over the theft of a female from a courting male by another male.

Courtships ended for a variety of reasons (Table 2.1). These include females leaving after being inside the well a male had taken her to, males leaving to engage in territorial activities, interference of other species, and success. Of 160 courtship bouts witnessed in 1999, 128 were "terminal," 32 "non-terminal."

Terminal courtship bouts are those that are not resumed within 15 minutes of interruption. Males leave non-terminal courtship bouts for territorial activities

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more often than they leave terminal courtship bouts for territorial activities.

Females, however, are equally likely to leave terminal and non-terminal bouts after being led inside a well by a male (Table 2.2).

In all instances in which a competitor species interrupted a courtship, the interruption terminated that courtship. The only heterospecific frog species seen to directly interfere with the courtships of M. laevigata was Plethodontohyla notostica. However, two other microhylids—Anodonthyla boulengeri and Platypelis grandis—also used wells, and prevented M. laevigata from using them.

Females oviposited in wells at different heights relative to the water line when they were engaged in courtship, versus when they were performing maternal care by depositing single eggs for tadpoles. During courtships, females oviposited above the water line 18 of 21 times, while during instances of maternal care, they deposited at or below the water line 4 out of 5 times (Fisher's

Exact Test, N=26, P ≤ 0.01). These data include only those ovipositions for which there was no ambiguity about the number or placement of resultant eggs. Of those same 21 non-ambiguous courtships observed in 1999 , mean clutch size was 1.29 ± 0.12, with a range of 1-3.

A larval crane fly (Limonia renaudi Alexander, Tipulidae) was observed eating the eggs of Mantella laevigata, as well as those of one of the other two species of frogs that breed in bamboo wells on Nosy Mangabe, Plethodontohyla notostica.

The tipulid larvae attach their mouth parts to the gelatinous capsule, and suck out the ovum. Embryos in later stages of development stop moving during egg predation, and are then eaten.

Tadpoles were never observed to engage in cannibalism. Up to four tadpoles of different developmental stages were observed coexisting in wells, and smaller

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tadpoles did not disappear from wells with larger tadpoles in them more often than they disappeared from wells in which they were the only inhabitants.

Males discriminate between wells on the basis of tipulid and heterospecific frog presence (Table 2.3). Wells to which males take females during courtships do not differ significantly from average viable wells with regard to tadpole presence. Wells to which males lead females tend to have more conspecific eggs in them than do random wells, but this is non-significant (Table 2.3). Females are not left with the option to discriminate between wells on the basis of heterospecific frog presence, as males never take them to these wells. A female rejected the one tipulid-filled well that a male took her to. Furthermore, there is a significant tendency for courted females to reject wells with tadpoles in them.

As with wells that males choose, wells that females accept have a non-significant tendency to contain more eggs than the average (Table 2.4.)

Most heterospecific frog species are avoided by M. laevigata when choosing oviposition sites, including the microhylids Anodonthyla boulengeri,

Plethodontohyla notostica, and Platypelis grandis. Platypelis grandis is a large, short- term resident that presents an obstacle for less than 24 hours. In contrast, the first two species move in and produce clutches, which the fathers stay with until metamorphosis, effectively removing those wells from use by M. laevigata for a month or more. In one instance, I observed a preexisting M. laevigata tadpole survive ten days in the same well with a Plethodontohyla notostica father and clutch. For over one month after the Plethodontohyla father abandoned the clutch, it decayed in the well while the M. laevigata tadpole was still developing in that well. The M. laevigata tadpole survived to metamorphosis. In several other cases, however, when Plethodontohyla notostica mating pairs moved in, and

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fathers stayed for over a month with their developing brood, previously existing

M. laevigata eggs or tadpoles were never seen again in those wells. The other paternal-care giving microhylid that usurps wells from M. laevigata, Anodonthyla boulengeri, is more common in forest than in bamboo stands, where the watched populations of M. laevigata live. Thus, no direct competition between A. boulengeri and M. laevigata for wells was seen, though A. boulengeri was observed occupying wells that M. laevigata previously had occupied. A. boulengeri and M. laevigata were both observed to move into artificial wells that were established in forest plots, suggesting possible competition for wells between these two species as well (see chapter three).

Not all heterospecific frog species are discriminated against by M. laevigata when choosing oviposition sites. Platypelis cowani, a small (25 mm SVL) microhylid species was often found in wells, then difficult to locate minutes later, when no frogs had been observed to leave. As such, no data were taken on P. cowani presence in wells due to suspected inaccuracies. However, in one directly observed M. laevigata courtship, there were two P. cowani easily seen inside the well in which the M. laevigata mating pair oviposited an egg.

Discussion

Mantella laevigata reveals a surprising number of behavioral convergences with the aposematic dendrobatids. The extended, repeated fights between males over territory, the stereotyped and highly tactile courtship sequences, and the

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maternal care that is given by females to tadpoles in the form of unfertilized eggs all resemble behaviors of some dendrobatids. Convergent morphology or behavior may provide information about the evolution of adaptations by revealing universal rules or causal chains. For instance: toxicity is a prerequisite for aposematic coloration; aposematic coloration leads to diurnality and clumping of individuals due to lower predation pressure; clumping allows for greater complexity of social interactions, which includes males trying to control females through controlling the resources they need access to (Emlen & Oring

1977; Heying 1997; Vences et al. 1998). Perhaps of more biological significance, however, are the differences between the dendrobatids and Mantella that emerge on closer inspection, and the specifics of the M. laevigata system.

Maternal care in M. laevigata is probably facultative, rather than obligate, given the small number of occurrences observed during this study. The evidence of maternal care may often disappear quickly, as hungry tadpoles eat trophic eggs within minutes of deposition. Most tadpoles were never seen receiving maternal care, which suggests that many tadpoles obtain nutrition from non- maternal sources. Whereas in most parental-care-giving dendrobatids, clutches are laid in one place, allowed to hatch, and the tadpoles are then transported, usually singly, to bromeliads or other wells (reviews in Weygoldt 1987; and

Crump 1995; but see Summers 1999 for an exception); in M. laevigata the eggs and tadpoles develop in the same space. Thus, courted females lay eggs while amplexed by males in the same phytotelmata used by lone females for maternal care when ovipositing for their already hatched offspring. These spaces are limiting for M. laevigata (chapter three), and males often take females to wells that already have developing eggs or tadpoles in them during courtships. If a

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male has fathered tadpoles currently developing in a well he actively defends, then the male's potential gain from convincing a female to oviposit in that well is increased. The egg the female lays during courtship may result in another offspring of his, or may go to feed his already existing offspring. Whether pre- zygotic or post-zygotic investment, his effort is parental in nature. This should be considered reproductive parasitism on the part of the male (Weygoldt 1987;

Summers 1999).

The “icebox hypothesis,” which postulates that some offspring provide indirect parental investment by providing food for other offspring, is predicted to be in effect in such a system, where future conditions are unpredictable (Mock

& Parker 1997). If heavy rains bring the water level in a well up to the level of an egg laid during courtship, that egg will be eaten if the well contains a tadpole. In such a case, however, that egg, left uneaten, would likely die, due to insufficient oxygen (Seymour & Bradford 1995). Females, then, might be expected to return to wells already containing their offspring for future courtships, as egg predation in such a well still furthers the female’s reproductive success. It is not clear that females are doing this, but males do lead females to defended wells during courtships that already contain the male’s tadpoles. Higher certainty of paternity than maternity in individual wells might have led to this manifestation of the icebox hypothesis, in which oviposition-site defending males are more eager than non-territorial females to have eggs laid in particular viable sites.

In response to males evolving reproductively parasitic behavior to further their reproductive success, females are expected to evolve detection mechanisms by which to avoid such parasitism during courtships. In M. laevigata, the evidence is strong that females actively discriminate against wells with tadpoles

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during courtships (Table 2.4). Tadpoles, in turn, are likely to evolve mechanisms by which to hide from females. The scouting behavior of females serves to give them multiple interactions with possible oviposition sites, such that when a female is taken to a well by a male during courtship, she already has some information about the quality and inhabitants of that well.

When courted females do oviposit into wells with tadpoles in them, the resultant egg (or occasionally eggs) is usually laid above the water line. Tadpoles are unable to reach eggs above the water line, except in rare cases where their forelimbs are well-developed but they have not yet dispersed from the well; thus the oviposition location is another mechanism by which females can attempt to protect their offspring from becoming food for another. Because wells are limiting, however, it is rare that M. laevigata tadpoles exist in wells without conspecific eggs on the walls of that well. When other courting pairs, or individuals of either sex who are scouting wells, move around in them, eggs are often dislodged into the water below. Thus, unlike the dendrobatid species with obligately oophagous tadpoles (e. g., Dendrobates pumilio, Brust 1993; and the other four species in the D. pumilio group, Crump 1995; and Dendrobates vanzolinii, Caldwell & de Oliveira 1999), tadpoles of M. laevigata have sources of conspecific eggs other than from their mothers. However, other species of dendrobatids have also been observed to cannibalize conspecific, fertilized eggs

(e.g., Dendrobates ventrimaculatus, Summers & Amos 1997; Summers 1999). All cases of maternal care observed in M. laevigata involved non-courted females and were directed at tadpoles in wells in which eggs had not been seen for at least three days, suggesting that females monitor the wells in which their tadpoles live. There is no evidence in M. laevigata that tadpoles cannibalize other tadpoles.

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In , small clutch size, large egg size, and terrestrial deposition of eggs are often correlated with a high degree of parental care in individual offspring (Crump 1995). M. laevigata usually exhibits the minimum possible clutch size, its ova are large at approximately 3-3.5 mm in diameter, and, though not having terrestrial oviposition per se, females oviposit on the sides of wells above the water line, which is presumably a defense against egg predation, like terrestrial oviposition. Given these predictors of parental care, facultative maternal care probably does not describe the extent of care in offspring in M. laevigata. Males may provide parental care that is not immediately identifiable as such. Males defend wells actively, often leaving individual courtships to chase other males away from their territories. Fights between M. laevigata males serve, most often, to maintain the territorial status quo. Initiators win fights most of the time, and initiators are usually resident males. (This is true across taxa in which it has been studied, e.g., Tasmanian hens, Putland & Goldizen 1998.)

Although non-resident males have been seen successfully mating in resident males' wells, this is the exception. A male “confident” of his paternity--which I cannot assess without molecular data--stands to increase his reproductive success through courtship regardless of the future of the egg that is laid.

Reproductive parasitism by a male to feed a pre-existing offspring is clearly parental investment on behalf of that offspring. Male defense of wells when tadpoles are inside could also be considered parental investment. If a male M. laevigata drives away individuals of one of the two paternal-care giving microhylid frog species from a well with his offspring, he has probably saved his tadpole's life. Individuals of both Plethodontohyla notostica and Anodonthyla boulengeri fill a well with their clutch and father, and remain there for more than

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a month, making it functionally impossible for M. laevigata to co-exist in that space. This anuran phytotelm community is ecologically quite similar to a neotropical phytotelm community which includes females of Dendrobates castaneoticus (which deposit a single tadpole into each phytotelm) and Bufo castaneoticus (which oviposit 61-387 eggs into each phytotome, Caldwell 1993).

Other competitors for well space include land crabs, which have, on occasion, been observed to move into wells and eat M. laevigata eggs. I predict that success of crabs moving into wells is diminished by the presence of an actively defending male, as only crabs already on the inside of a well interacted with adult M. laevigata.

Courtships are the most direct route to reproductive success that these frogs have, but courtships end frequently without an egg being laid (Tables 2.1, 2.2).

This raises the question: what is more important to long-term reproductive success than the continuation of the present courtship? Females most frequently leave courtships only after investigating the well into which the male expects her to oviposit, suggesting that some measure of well quality that she is gauging is a better predictor of offspring success than the particular male with which she is in courtship. Sexual selection experiments support this hypothesis (see chapter five).

When males leave courtships, however, more explanation is required. Males rarely leave to court other females, even though the quality of the tadpole in the well a male defends must be critical to the father's reproductive success. Thus, males do not appear to exhibit mate choice in this species, although some parameters of the social system fit predictions of male mate choice: males provide benefits to both females and offspring by defending oviposition sites,

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but the courtship process is long enough to limit, though not preclude, multiple matings by males (Krupa 1995). Furthermore, the resource provided by the male in this system cannot be used simultaneously by a large number of offspring, as tadpoles will eat younger, conspecific eggs if they can. “Mate” choice for oviposition sites rather than individuals is consistent with experimental evidence that these sites are limiting for M. laevigata (see chapter three).

Males determine the quality of wells before they begin courting females, which is consistent with focal observations of males scouting the area and investigating various wells. Thus, before females have a chance to reject wells, males have already surveyed wells, and do not take potential mates to wells containing crane-fly larvae or members of other frog species (Table 2.3). This is not due to different requirements for the various species, as the same wells discriminated against by M. laevigata when they contain other frogs or tipulid larvae are used for reproduction when they are free of competitors and predators. In the first reported system in which frogs assess oviposition sites for egg density—Hyla pseudopuma, a tropical hylid with known egg cannibalism by tadpoles--females and males are both choosy with regard to the phytotelmata in which they oviposit (Crump 1991). Findings were similar in three dendrobatid systems. Summers observed male Dendrobates auratus exploring oviposition sites in succession before taking tadpoles to them (Summers 1989). Caldwell hypothesized that adult D. castaneoticus carrying tadpoles detect predatory insect larvae in phytotelmata, and discriminate against wells containing them

(Caldwell 1993). Finally, Summers demonstrated that D. ventrimaculatus males do discriminate against pools containing large tadpoles when courting females and when depositing tadpoles (Summers 1999).

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Though the evidence is strong that M. laevigata discriminate against wells with crane-fly larvae in them (Table 2.3), M. laevigata males cannot prevent adult crane-flies from ovipositing in wells that already contain frog eggs. Because clutch size is minimal in M. laevigata, the effect of a single voracious crane-fly larva on the mating effort of M. laevigata will potentially be much greater than that on a clutch of Plethodontohyla notostica eggs (n > 40 in all clutches observed).

Minimal clutch size may, however, be due in part to a selective response to tipulid presence, such that females scatter their eggs across multiple wells in order to reduce the likelihood of all of their offspring being eaten in a single predation event.

I predict that inter-clutch kin selection for accelerated hatching will occur when an egg inhabiting the same well is attacked by a crane-fly larva. This follows from Warkentin’s work on red-eyed tree frogs, in which she found that eggs within a clutch that is being attacked by an egg-eating snake escape snake predation by hatching early and dropping into the water below (Warkentin

1995). Due to selection pressure to detect conspecific eggs being eaten, I hypothesize that the destruction of M. laevigata eggs by crane-fly larvae accelerates the hatching of conspecific eggs in the same well, regardless of relationship. Although other M. laevigata eggs in the same well are usually not full siblings, they are often, if not always, half-sibs, the parent in common being the male defending the site. The prediction of such adaptive plasticity in M. laevigata eggs differs from Warkentin’s system primarily in that the advantage to early hatching would go to half-siblings of the predated egg, rather than members of the same clutch.

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Though M. laevigata and P. notostica eggs are both at risk from crane-fly larvae, tadpoles do not appear to be, as they were frequently seen co-existing in wells with crane-fly larvae. In such cases, the crane-fly larvae were often positioned above the water line, suggesting a possible reverse predation by tadpoles on the crane-flies, though this has not been tested.

Several aspects of the reproductive behavior of M. laevigata are clearly context-dependent. Examples include males scouting for oviposition sites, and not taking females to previously favored wells which are now changed for the worse, due to new inhabitants or low water level; females both scouting for wells alone, and discriminating between wells at the time of courtship, depending on the current contents of the well; and females ovipositing at different positions relative to the water line when they are in courtship, versus providing maternal care. The assessment required for all of these choices points to a sophisticated set of standards and rules on which choices are based.

To summarize, males defend water-filled wells, which are used as oviposition sites by females. Extremely low clutch size suggests that rare instances of direct maternal provisioning of tadpoles are not the only form of parental care in this species. Males win, reproductively, whenever they court a female that oviposits in a well in which one of the male's offspring already exists. A father’s reproductive success is aided whenever his tadpole eats an egg, be it from the tadpole's mother, the result of a courtship involving the father, or the result of a courtship involving neither mother nor father. Males do not take females to wells that contain frog species larger than themselves, nor do they expose courted females to the predatory crane-fly larvae that eat frog eggs. Females avoid courtships in wells that already have tadpoles in them, but tadpoles are

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probably selected to hide from disturbance, so as to increase their chances of being provisioned. The previously unknown social system and well community of Mantella laevigata is as complex and evolutionarily fascinating as that of the better-studied dart-poison frogs.

Acknowledgments

I am indebted to Jessica Metcalf and Glenn Fox for helping to collect focal observations, and to Bret Weinstein for intellectual support and additional field assistance. I am grateful to Kyle Summers, Marty Crump, Arnold Kluge, Richard

Alexander, Barbara Smuts and Ron Nussbaum for comments on earlier drafts of this paper, and to two anonymous reviewers. John Megahan provided the illustration of chinning behavior. Kathy Welch was of great help with statistical consultation. This work would not have been possible without the support of the government of Madagascar; logistical support in country was given by Ron

Nussbaum, WCS, Projet Masoala, and MICET. This research was supported by grants from Animal Behaviour, Sigma Xi, The University of Michigan Museum of Zoology, Department of Biology, and Rackham Graduate School, and the

Sokol International Research Fellowship. A slightly modified version of this chapter is in press at Animal Behaviour (manuscript #A8794).

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Table 2.1: How Courtship Bouts End

% of subset Total Frequency Reason for Courtship Termination (Female left, (% of total bouts) or Male left)* Female left 52 (32.5%) after being inside a well 31 (19.4%) 59.6% for another male 3 (1.9%) 5.8% Male left 61 (38.1%) to initiate territorial defense 27 (16.9%) 44.3% to defend against another male 14 (8.8%) 23.0% for another female 3 (1.9%) 4.9% Competitor interference (P. notostica, crab) 6 (3.8%) Success (oviposition) 21 (13.1%)

Courtship bouts (N=160) end in a variety of ways, including females or males abandoning the courtship for any of several reasons. Frequency does not total 100% because it was sometimes impossible to gauge why or how a courtship ended.

*The final column is a measure of the conditions under which males and females left courtships (e.g., Of those females who left courtships, 59.6% (31/52) left only after being inside a well, while only 5.8% (3/52) left for another male).

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Table 2.2: Differences in Courtship Termination between Terminal and Non-Terminal Bouts

Non-Terminal Terminal Difference Termination Reason Bouts (%) Bouts (%) (p value) Female leaves after being A 7/32 (21.9) 24/128 (18.75) 0.65 inside well Male leaves to initiate B 10/32 (31.3) 17/128 (13.3) 0.02 territorial defense Male leaves to defend C 6/32 (18.8) 8/128 (6.3) < 0.0001 himself against another male Male leaves to engage in D 16/32 (50.0) 25/128 (19.6) 0.001 territorial activity (B + C)

P-values generated using Logistic Regression with clustering of females (A) or males (B-D).

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Table 2.3: Male Discrimination of Wells in Bamboo Stand 4

Number of wells containing other organisms Type of well Other frog Eggs Tadpoles Tipulids species* 71 90 43 71 Average Well (N=238) (29.8%) (37.8%) (29.8%) (29.8%) Wells Males take 13 15 1 0 Females to (N=31) (41.9%) (48.4%) (3.2%) (0%) Difference (Chi-Square Goodness p=0.11 p=0.27 p=0.02 p=0.0004 of fit p-value)

*Other frog species include only Plethodontohyla notostica and Anodonthyla boulengeri

"Average Wells" include all data from wells in stand 4, which were surveyed every three days independent of focal watches. Each well was included in the survey only if, at some point during the 3.5 months of survey, evidence of M. laevigata reproductive activity was observed in that well (eggs, tadpoles, or courting adults directly observed).

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Table 2.4: Female Discrimination of Wells during Courtships

Egg Tadpole Type of well Occupied Occupied Wells Males take Females to (N=31) 13 (41.9%) 15 (48.4%)

Wells Females Accept (N=18) 9 (50.0%) 6 (33.3%) Difference p=0.12 p=0.02 (Logistic regression 2-tailed p-values)

Females reject wells which are already tadpole-occupied for oviposition. They do not discriminate against egg presence in wells; rather, there is a non-significant trend for females to favor wells with eggs in them.

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Figure 2.1: Map of Bamboo Stands containing Focal Populations on Nosy Mangabe

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Figure 2.2: Chinning Behavior of Male Mantella laevigata during Courtship

During both initiation and continuation of courtship, the male repeatedly makes contact with the female by resting his throat on either the top of her head, or her back, while softly emitting a courtship call. The relative position of the courting animals is variable, but usually the male is facing the female, or perpendicular to her.

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Figure 2.2: Chinning Behavior of Male Mantella laevigata during Courtship